Lv shsirt3

MNK3 cells were cultured in a cell medium composed of DMEM, 10% FBS, and penicillin/streptomycin. For the construction of the SIRT3 knockdown cell line, MNK3 cells were co-cultured with SIRT3 knockdown lentivirus (LV-shSIRT3) or blank vector (LV-shNC) (Shanghai Genechem Co., Ltd, Shanghai, China.) for 12 h, and then the culture medium was replaced. The lentivirus-infected cells were screened in a culture medium containing puromycin for purification. To detect the effect of DHM and palmitic acid (PA) on MNK3 cells, cells were pretreated with DHM for 4 h followed by PA (0.2 mM) for another 12 h. To detect the involvement of AMPK, SIRT3, or STAT3 in DHM-induced benefits on MNK3 cells, cells were pretreated with inhibitors of SIRT3 (3-TYP, 50 μM; MCE, Newark, USA), AMPK (Dorsomorphin, 10 μM; MCE) or STAT3 (Static, 5 μM; Selleck Chemicals, TX, USA) for 2 h prior to the treatment of DHM and PA.

Lentivirus-mediated SIRT3 overexpression (Lv-SIRT3) and lentivirus-mediated SIRT3 knockdown (Lv-shSIRT3) constructs were synthesized by Genechem (Shanghai, China). ASCs of third passage were cultured in 6-well plates in GM at an initial density of 1.2 × 10⁶/well. When the cells reached about 70% confluency, ASCs were transfected with the constructed lentivirus harboring rat SIRT3 shRNA, the SIRT3-coding sequence, or GFP-expressing control vector (Genechem, Shanghai, China) at a multiplicity of infection (MOI) of 20. After incubation with the above constructs for 8-12 h, the medium was replaced by GM for another 72 h culture. For the transient knockdown of PGC-1α, PGC-1α-siRNA and negative control-siRNA (NC-siRNA) were designed and synthesized by GenePharma Corporation (Shanghai, China), the transfection was performed by using LipoHigh reagent (Sangon Biotech, China), with LipoHigh : siRNA = 5 μI : 100 pmol for each well of 6-well plates. Transfection efficiencies of SIRT3 and PGC-1α were confirmed with Western blot and real-time PCR.